foxm1-mut  (Addgene inc)

Journal: Investigative Ophthalmology & Visual Science

Article Title: ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation

doi: 10.1167/iovs.65.12.34

Figure Lengend Snippet: ALKBH5 mediates m6A modification of FOXM1. ( A ) The qRT–PCR analysis of FOXM1 in the control group, IL-6 group, IL-6 + siNC group, and IL-6 + siALKBH5 group, with the fold ratio of the control group normalized to 1. ( B ) Western blot analysis ( left graph ) and quantitative analysis ( right graph ) of FOXM1 protein levels in four groups, with the fold ratio of the control group normalized to 1. ( C ) The SRAMP online tool identified the m6A modification site on FOXM1 mRNA. The prediction results were sorted by position on the horizontal axis, and the vertical axis showed the combined prediction scores by three random forest classifiers. ( D ) The schematic of the most likely binding site generated by SRAMP. ( E ) MeRIP–qPCR analysis of FOXM1 m6A enrichment in normal and CNV corneas, with the fold ratio of the negative control normalized to 1. ( F ) MeRIP–qPCR analysis of FOXM1 m6A enrichment in IL-6-induced HUVECs with or without ALKBH5 knockdown, with the fold ratio of the negative control normalized to 1. ( G ) The interaction between ALKBH5 and FOXM1 using the dual-luciferase reporter assay in IL-6-induced HUVECs with or without ALKBH5 knockdown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, no significant difference.

Article Snippet: Customized FOXM1-WT and FOXM1-MUT constructs were purchased from GeneChem (Shanghai, China).

Techniques: Modification, Quantitative RT-PCR, Control, Western Blot, Binding Assay, Generated, Negative Control, Knockdown, Luciferase, Reporter Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation

doi: 10.1167/iovs.65.12.34

Figure Lengend Snippet: FOXM1 overexpression reverses ALKBH5 depletion effects. ( A ) Western blot analysis ( leftmost image ) and quantitative analysis ( right two images ) of ALKBH5 and FOXM1 protein levels, with the fold ratio of the control group normalized to 1. ( B ) The CCK8 assay was used to analyze the cell viability of HUVECs. ( C, D ) The images taken at 0 and 24 hours after scratching in the wound healing assay and the assessment of wound healing rates. Scale bar: 200 µm. ( E ) The formation of tubes was visualized after cell seeding on Matrigel and incubated for four hours. Scale bar: 100 µm. Quantification results of capillary lengths ( F ) and branch points ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, no significant difference.

Article Snippet: Customized FOXM1-WT and FOXM1-MUT constructs were purchased from GeneChem (Shanghai, China).

Techniques: Over Expression, Western Blot, Control, CCK-8 Assay, Wound Healing Assay, Incubation

Journal: Oncotarget

Article Title: Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression

doi: 10.18632/oncotarget.18507

Figure Lengend Snippet: (A) qRT-PCR. HUVECs were grown and transfected with MALAT1 siRNA (#1 and #2) or scrambled siRNA and then subjected to qRT-PCR analysis of MALAT1 expression (n=3). (B) and (C) Cell proliferation assay. HUVECs were grown and transfected with MALAT1 siRNA or scrambled siRNA and then subjected to the cell proliferation assay. The graph in B is quantified data from C. (D) qRT-PCR. HUVECs were grown and transfected with MALAT1 siRNA (#1) or scrambled siRNA and then subjected to qRT-PCR analysis of FOXM1 expression (n=3). (E) Western blot. HUVECs were grown and transfected with MALAT1 siRNA or scrambled siRNA and then subjected to immunoblotting analysis of FOXM1 expression.

Article Snippet: For the dual luciferase reporter assay, HEK293T cells were co-transfected with 150 ng of a firefly luciferase reporter plasmid pGL3-MALAT1-Seed1 (Seed1), or pGL3- MALAT1-Seed2 (Seed2) and a renilla luciferase vector (pRL-SV40, Promega) plus small RNAs (miR-101 mimics or negative control RNAs) using Lipofectamine 2000 (Invitrogen). pGL3-FOXM1-UTR-wt and pGL3-FOXM1-UTR-mut (Sangon Biotech, Shanghai, China) were also transfected into HEK293T cell using Lipofectamine 2000.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Proliferation Assay, Western Blot

Journal: Oncotarget

Article Title: Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression

doi: 10.18632/oncotarget.18507

Figure Lengend Snippet: (A) qRT-PCR. HUVECs were grown and transfected with FOXM1 or Lv-control (lentivirus-control), shRNA or scrambled shRNA and then subjected to qRT-PCR analysis of FOXM1 expression. (B) Western blot. HUVECs were grown and transfected with FOXM1 shRNA or scrambled siRNA and then subjected to immunoblotting analysis of FOXM1 expression. (C) and (D) Cell proliferation assay. HUVECs were grown and transfected with FOXM1 shRNA or scrambled shRNA and then subjected to the cell proliferation assay. The graph in C is quantified data from D.

Article Snippet: For the dual luciferase reporter assay, HEK293T cells were co-transfected with 150 ng of a firefly luciferase reporter plasmid pGL3-MALAT1-Seed1 (Seed1), or pGL3- MALAT1-Seed2 (Seed2) and a renilla luciferase vector (pRL-SV40, Promega) plus small RNAs (miR-101 mimics or negative control RNAs) using Lipofectamine 2000 (Invitrogen). pGL3-FOXM1-UTR-wt and pGL3-FOXM1-UTR-mut (Sangon Biotech, Shanghai, China) were also transfected into HEK293T cell using Lipofectamine 2000.

Techniques: Quantitative RT-PCR, Transfection, shRNA, Expressing, Western Blot, Proliferation Assay

Journal: Oncotarget

Article Title: Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression

doi: 10.18632/oncotarget.18507

Figure Lengend Snippet: (A) Gene Ontology classification of the predicted miR-320a target genes after integration of four algorithms (Targetscan, TarBase, miRDB, and microRNA.org ). (B) The GO classification of miR-320a targets predicted by integration of the four algorithms. (C) qRT-PCR. HUVECs were grown and transfected with miR-320a mimics or NC and then subjected to qRT-PCR analysis of FOXM1 expression. (D) Western blot. HUVECs were grown and transfected with miR-320a mimics or NC and then subjected to Western blot analysis of FOXM1 expression. (E) Prediction of the miR-320a binding sites to the FOXM1 3’-UTR using the sequence complementarity and phylogenic conservation of 7/8-nucleotide seed sequence prediction. (F) Luciferase reporter assay. The relative luciferase activity mediated by the reporter constructs harboring the wild type (wt) or mutated FOXM1 3’-UTR upon transfection with miR-mimics NC or miR-320a mimics.

Article Snippet: For the dual luciferase reporter assay, HEK293T cells were co-transfected with 150 ng of a firefly luciferase reporter plasmid pGL3-MALAT1-Seed1 (Seed1), or pGL3- MALAT1-Seed2 (Seed2) and a renilla luciferase vector (pRL-SV40, Promega) plus small RNAs (miR-101 mimics or negative control RNAs) using Lipofectamine 2000 (Invitrogen). pGL3-FOXM1-UTR-wt and pGL3-FOXM1-UTR-mut (Sangon Biotech, Shanghai, China) were also transfected into HEK293T cell using Lipofectamine 2000.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Construct

Journal: Oncotarget

Article Title: Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression

doi: 10.18632/oncotarget.18507

Figure Lengend Snippet: (A) qRT-PCR. HUVECs were grown and transfected with MALAT1 siRNA, miR-320a inhibitors, or their combination and then subjected to qRT-PCR analysis of FOXM1 expression. (B) Western blot. HUVECs were grown and transfected with MALAT1 siRNA, miR-320a inhibitors, or their combination and then subjected to Western blot analysis of FOXM1 expression. (C) and (D) Cell proliferation assay. HUVECs were grown and transfected with MALAT1 siRNA, miR-320a inhibitors or their combination and then subjected to cell proliferation assay.

Article Snippet: For the dual luciferase reporter assay, HEK293T cells were co-transfected with 150 ng of a firefly luciferase reporter plasmid pGL3-MALAT1-Seed1 (Seed1), or pGL3- MALAT1-Seed2 (Seed2) and a renilla luciferase vector (pRL-SV40, Promega) plus small RNAs (miR-101 mimics or negative control RNAs) using Lipofectamine 2000 (Invitrogen). pGL3-FOXM1-UTR-wt and pGL3-FOXM1-UTR-mut (Sangon Biotech, Shanghai, China) were also transfected into HEK293T cell using Lipofectamine 2000.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Western Blot, Proliferation Assay

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: FOXM1 is SUMOylated by SUMO1. ( a ) Protein lysates were prepared from MCF-7 cells with or without transfection of the SUMO E2-ligase Ubc-9. FOXM1 was detected by western blot analysis and a higher molecular weight form was observed consistent with SUMOylation of FOXM1. ( b ) MCF-7 cells were transfected with FOXM1, Ubc9 or SUMO1, -2 or -3, and FOXM1 was detected by western blot analysis. Multiple higher molecular weight forms of FOXM1 were observed, consistent with poly-SUMOylation. ( c ) MCF-7 cells were co-transfected with His-SUMO1, FOXM1 and Ubc9. SUMOylated proteins were purified using Ni 2+ -column affinity pulldown under denaturing conditions (8 M urea). Input and His-tagged proteins were probed for FOXM1, and poly-SUMOylated FOXM1 was detectable demonstrating the covalent linkage of His-SUMO1 to FOXM1. ( d ) IPTG-inducible recombinant His-tagged FOXM1 was expressed in E. coli and isolated using Ni 2+ -column affinity pulldown. Cell lysates from induced and non-induced E. coli were incubated with Ubc9 and SUMO1 in the presence and absence of Mg 2+ -ATP and reaction buffer, and resolved using SDS-PAGE. SUMO1 was detected by western blot analysis. ( e ) MCF-7 cells were transfected with constructs encoding FOXM1 fused to mouse Ubc9 (FOXM1-Ubc9) with or without eGFP-tagged SUMO1. The higher molecular weight band detected by FOXM1 and Ubc9 antibodies, which is consistent with the auto-SUMOylation of FOXM1-Ubc9, is indicated. SUMOylation was enhanced and increased in molecular weight by co-transfection of eGFP-SUMO1 (indicated by the multiple arrows). ( f ) MCF-7 cells were co-transfected with FOXM1, SUMO1, Ubc9, SENP-1 and the dominant-negative SENP-1 construct (SENP1-C630S), and lyssates were analysed by western blot analysis.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Transfection, Western Blot, Molecular Weight, Purification, Affinity Column, Recombinant, Isolation, Incubation, SDS Page, Construct, Cotransfection, Dominant Negative Mutation

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: FOXM1 is SUMOylated in response to epirubicin treatment. ( a ) MCF-7 cells were treated with epirubicin (1 μ M ) for 0, 6 and 24 h. Co-immunoprecipitation (co-IP) was performed with a FOXM1 antibody and was probed for SUMO1 and vice versa ; inputs (1/10 of IP) and IP products with IgG and specific antibodies were resolved on western blot analysis and were probed for FOXM1 and SUMO1. High molecular weights of FOXM1- and SUMO1-containing species are highlighted ‘*'. ( b ) MCF-7 cells co-transfected with 0.05 μg of eGFP-FOXM1 and 0.025 μg per well of either tRFP-SUMO1 (FRET acceptor) or empty expression plasmids were treated with 0.1 μ M epirubicin for 0, 2, 4, 6 and 24 h. Intensity-merged fluorescence lifetime images of doxorubicin treatment time course are shown. The reduction in donor fluorescence lifetime indicates the occurrence of FRET, implying that eGFP-FOXM1 and tRFP-SUMO1 are within 10 nm of each other. ( c ) Analysis of fluorescence lifetime data, showing mean lifetimes ±s.e.m. of eGFP-FOXM1 fluorescence,was determined by fitting the fluorescence decay as described. Each measurement is based on >100 cells. The results show that eGFP-FOXM1 and tRFP-SUMO1 interactions significantly increase after 6 or 8 h epirubicin treatment (Tukey's HSD test: http://www.amazon.co.uk/Biostatistics-Methodology-Sciences-Probability-Statistics/dp/0471031852 : 0 versus 3, 6, 8 or 24 h; *significant P <0.05; NS, not significant).

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Fluorescence

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: SUMOylation of FOXM1 occurs during mitotic arrest. ( a ) MCF-7 cells were treated with dimethyl sulfoxide (DMSO) (0.001% (v/v); 16 h) or nocodazole (0.0001 mg/ml; 16 h), and immunoprecipitation (IP) was performed with a FOXM1 antibody; inputs and IP products were resolved on western blot analysis and were probed for SUMO1. The membrane was then reprobed with a FOXM1 antibody. Arrows indicate higher molecular weight forms of FOXM1. ( b ) MCF-7 cells were treated with paclitaxel (100 n M ) for 0, 6 and 24 h. Co-IP was performed with a FOXM1 antibody and was probed for SUMO1 and vice versa ; inputs (1/10 of IP) and IP products with IgG and specific antibodies were resolved on western blot analysis and were probed for FOXM1 and SUMO1. * indicates higher molecular weight FOXM1 complex.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Immunoprecipitation, Western Blot, Molecular Weight, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: FOXM1 is SUMOylated at multiple sites. ( a ) Schematic showing consensus SUMOylation sites in FOXM1 identified using online computational prediction software Abgent SUMOplot (Abgent, Maidenhead, UK) and SUMOsp 2.0 (The Cuckoo Workgroup, Hefei, China). NRD, N-terminal regulatory domain; FKH, forkhead domain; TAD, transactivation domain. ( b ) Site-directed mutagenesis was performed using WT-FOXM1 expression vector at the indicated sites (lysine to arginine). In all cases, mutants were confirmed by sequencing. MCF-7 cells were then co-transfected with WT- or indicated mutant-FOXM1, SUMO1 and Ubc9 constructs, and 24 h later protein lysates were prepared and FOXM1 SUMOylation was determined by western blot analysis. ( c ) FOXM1 mutants were generated, containing multiple lysine-to-arginine mutations, from FOXM11X(K>R) (K210R) to FOXM17X(K>R) (K201R/K218R/K356R/K440R/K460R/K478R/K495R). Mutational order was based on site location. FOXM1 mutant SUMOylation was determined as above. ( d ) FOXM17X(K>R) was subjected to reversal of individual mutations (R>K) to examine site redundancy. Mutants were assessed for SUMOylation as above and K356R and K440R were identified as redundant mutations. ( e ) MCF-7 cells were transfected with WT-FOXM1 or FOXM15X(K>R) (K201R/K218R/K460R/K478R/K495R) with or without Ubc9 and SUMO1. SUMOylation of FOXM1 was determined as above. ( f ) MCF-7 cells were transfected with either FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 with or without eGFP-tagged SUMO1. Loss of the auto-SUMOylated form of FOXM1 was observed in FOXM15X(K>R)-Ubc9. SUMOylation of FOXM1 was determined as above.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Software, Mutagenesis, Expressing, Plasmid Preparation, Sequencing, Transfection, Construct, Western Blot, Generated

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: SUMOylation of FOXM1 promotes its cytoplasmic localization and occurs preferentially on phosphorylated FOXM1. ( a ) MCF-7 cells were co-transfected with FOXM1, SUMO1 and Ubc9 constructs and cytoplasmic and nuclear fractions were isolated. FOXM1 SUMOylation and cell fractionation were confirmed by western blot analysis against FOXM1, SUMO1, lamin and β-tubulin. ( b ) MCF-7 cells were transfected with FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 and were fractionated as above. The cytoplasmically localized auto-SUMOylation event is indicated. ( c ) MCF-7 cells were transfected with FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 and stained for FOXM1, β-tubulin and 4′,6-diamidino-2-phenylindole before analysis by confocal microscopy. Images are representative of three independent experiments. ( d ) MCF-7 cells were transfected with FOXM1 or the N-terminal truncated FOXM1 (Δ-FOXM1) and cells were fractionated as above. ( e ) MCF-7 cells were co-transfected with WT-FOXM1 or mutant-FOXM15X(K>R) with or without SUMO1 and Ubc9 constructs, and 24 h later protein lysates were subjected to immunoprecipitation (IP) with a FOXM1 antibody. Precipitated proteins were separated by SDS-PAGE and FOXM1 phosphorylation was detected using a phosphorylated-M-phase-associated protein-specific antibody (MPM-2). The membrane was then reprobed with a FOXM1 antibody, demonstrating that the free FOXM1 displayed no MPM-2 detectable phosphorylation following SUMOylation in the WT-FOXM1, whereas the SUMOylated form of FOXM1 was detectable by MPM-2. These are indicated by boxes.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Transfection, Construct, Isolation, Cell Fractionation, Western Blot, Staining, Confocal Microscopy, Mutagenesis, Immunoprecipitation, SDS Page

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: SUMOylation of FOXM1 promotes its degradation and a SUMOylation mutant is resistant to Cdh1-mediated degradation. ( a ) MCF-7 cells were transfected with FOXM1 with or without Ubc9 and SUMO1, and 16 h later cells were treated with cycloheximide (CHX) or vehicle (0.001% (v/v) dimethyl sulfoxide (DMSO)) and protein lysates were prepared from 0 to 8 h following CHX treatment. Densitometry was used to quantify the unconjugated FOXM1 relative to the SUMOylated-FOXM1, for which independent background readings were taken. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. Densitometry was used to quantify FOXM1 levels and were normalized to β-tubulin. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. ( t -test: *significant P <0.05; otherwise not significant). ( b ) Asynchronous MCF-7 cells were co-transfected with FOXM-1 or FOXM15X(K>R) with or without increasing levels of Cdh1(4a). At 24 h after transfection, protein lysates were prepared and FOXM1 was examined by western blot analysis. The arrow indicates the higher molecular weight form of FOXM1 suggesting protein ubiquitination. ( c ) MCF-7 cells were transfected with FOXM1 or FOXM1(5XK>R) with or without Cdh1(4a), and 16 h later cells were treated with cycloheximide or vehicle (0.001% (v/v) DMSO) and protein lysates were prepared from 0 to 8 h following cycloheximide treatment. Densitometry was used to quantify FOXM1 levels and were normalized to β-tubulin. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. ( t -test: *significant P <0.05; otherwise not significant). ( d ) MCF-7 cells were co-transfected with FOXM1 or FOXM15X(K>R) with Flag-Cdh1(4a), and 24 h later protein lysates were subjected to co-immunoprecipitation (co-IP) with a FOXM1 antibody. Precipitated proteins were separated by SDS–PAGE and Flag-Cdh1(4a) binding was detected using an anti-Flag antibody. The membrane was then reprobed with a FOXM1 antibody. Ubiquitinated-FOXM1 (Ubq-FOXM1) and IgG are indicated by arrows. ( e ) MCF-7 cells were co-transfected with FOXM1 or FOXM15X(K>R) with or without haemagglutinin-ubiquitin (HA-Ubq). After 24 h, protein lysates were subjected to IP with a FOXM1 antibody. Precipitated proteins were separated by SDS–PAGE and ubiquitination of FOXM1 was detected by anti-HA antibody. The membrane was then reprobed with a FOXM1 antibody.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Mutagenesis, Transfection, Western Blot, Molecular Weight, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Binding Assay

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: SUMOylation of FOXM1 represses its transcriptional activity and a SUMOylation-deficient FOXM1 mutant can enhance cell proliferation. MCF-7 cells were co-transfected with FOXM1-Ubc9 or FOXM1(5XK>R)-Ubc9 (0–20 ng) and luciferase reporters were driven by ( a ) the FOXM1 6X-DNA-binding element (6XDBE), ( b ) the wild-type cyclin B1 promoter (cyclin B1-pGL2) or the cyclin B1 promoter containing mutations in the three consensus forkhead binding sites ( cyclin B1 -mut3-pGL2), or ( c ) the GADD45 promoter (GADD45-pGL2). After 8 h, cells were placed in 0.5% fetal calf serum for 24 h before the luciferase assay was performed. All DNA concentrations were normalized using empty vector. Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. ( d ) MCF-7 cells were transfected with FOXM1 with or without SUMO1. After 20 h, cells were treated with BrdU (10 μmol/l) for 4 h and then harvested and stained with propidium iodide and fluorescein isothiocyanate-conjugated anti-BrdU antibody and analysed by fluorescence-activated cell sorter (FACS) (20 000 gated events were counted; data are representative of three independent experiments; graph shows mean of three experiments±s.e.m.). ( e ) MCF-7 cells were transfected with empty vector, FOXM1 or FOXM15X(K>R), and 20 h later they were treated with BrdU for 4 h before being harvested and stained with propidium iodide- and fluorescein isothiocyanate-conjugated anti-BrdU. Fluoresence was determined by FACS analysis (20 000 gated events were counted; data are representative of three independent experiments; graph shows mean of three experiment ±s.e.m.) ( t -test: *significant P <0.05; otherwise not significant).

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Activity Assay, Mutagenesis, Transfection, Luciferase, Binding Assay, Plasmid Preparation, Staining, Fluorescence

Journal: Oncogene

Article Title: SUMOylation inhibits FOXM1 activity and delays mitotic transition

doi: 10.1038/onc.2013.546

Figure Lengend Snippet: SUMOylation of FOXM1 delays mitotic progression and deregulates cyclin B1 expression. ( a ) RPE-hTERT-GFP-α-tubulin cells were transfected with empty vector, pmCherry plus FOXM1-Ubc9 or pmCherry plus FOXM15X(K>R)-Ubc9 and synchronized in the G1 phase by double thymidine block. Following removal of thymidine, progression through mitosis was monitored by time-lapse phase-contrast and fluorescent microscopy. ( b ) Graphical representation of mitotic progression. Time from mitotic entry to exit, mitotic entry to metaphase alignment and metaphase alignment to anaphase onset were calculated on a total of 200 cells per condition from three independent experiments (mean±s.e.m.). Only cells that were transfected with the pmCherry-tagged FOXM1 constructs were included. ( c ) HeLa cells were transfected with empty vector, FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9, and 24 h later they were synchronised by double thymidine block. Following release from thymidine block, protein lysates were prepared from 0 to 13 h; lysates were resolved by gel electrophoresis and western blot analysis was performed for mitotic and S-phase-associated cyclins and checkpoint protein. β-Actin was used as a loading control; data are representative of three independent experiments.

Article Snippet: For the mitosis studies the FOXM1-Ubc9 and the FOXM1-mut-Ubc9 plasmids were subcloned into pmCherry plasmid (Invitrogen).

Techniques: Expressing, Transfection, Plasmid Preparation, Blocking Assay, Microscopy, Construct, Nucleic Acid Electrophoresis, Western Blot